Sulfhydryl tests and compounds therefor



United States Patent Ofiice Patented Jan. 23, l94

3,11%,663 SULFHYDRYL TESTS AND COMPQUNDS THEREFQR George L. Elhnan,Tiburon, Calih, assignor to The Dow Qhemical Company, Midland, Mich, acorporation of Delaware No Drawing. Filed Ilan. 26, 1959, Ser. No.788,735 2 filaims. ((Il. 23--23t)) The present invention relates tochemical analysis and is particularly directed to novel methods for thequalitative or quantitative determination of sulfhydryl groups, and tochemical compounds to be employed as reagents in the conduct of suchtests.

According to the present invention it has been discovered that abrilliant yellow color is produced when a composition containingsulfhydryl groups, that is mercaptoor thiol groups, corresponding to theformula is brought into contact with the test reagentbis-(4-nitrophenyl)-disulfide, or any of a very large number of testreagents having a bis-(substituted-4-nitrophenyl)disulfide structure.The said yellow color is of an intensity proportional to, among otherthings, the concentration of sulfhydryl groups present in the saidcomposition. The compounds to be used as reagents in the conduct of thesulfhydryl test of the present invention contain a structurecorresponding to the skeletal formula and include compounds havingsubstituents at any of the positions orthoand metato the disulfidelinkage, subject only to the limitations that any substituent in ringpositions metato a nitro group is non-nucleophilic, except that carboxylgroups and salts thereof may ocour in such position, and any branchingsubstituent in such position must have its branching moieties separatedfrom the benzene nucleus by at least one bridging atom. Nucleophilicradicals which are non-admissible in the said meta-positions and are,therefore, to be avoided in such position, accord with those set forthin Introduction to Theoretical Organic Chemistry, by Hermans (firstEnglish edition), Elsevier, 1954, p. 254 and following.

It will be seen that all the compounds thus suggested to be used in thepresent test retain the group or nucleus corresponding to the skeletalformula hereinbefore set forth, and it has been learned that compoundswhich contain this characteristic group are of value for the analyticaltest of the present invention, subject only to the limitations abovestated. Examples of substituents which may permissibly or advantageouslyoccur in one or more of the positions orthoto the disulfide linkage,that is to say, positions 2,6,2 and/or 6 in the skeletal formula, are astraight chain alkyl radical containing from 1 to 18 carbon atoms, abranched-chain organic radical of which the branching nearest a benzenemoiety of the said skeletal formula is separated therefrom by at leastone atom, an alkoxy radical of which the alkyl moiety contains from 1 to18 carbon atoms, phenyl, alkylphenyl, phenalkyl whereof the alkylenemoiety is a normal alkylene moiety and contains from 1 to 18 carbonatoms, and phenoxy. Examples of electronegative, that is to say,nucleophilic, substituents which should be avoided in the positionsorthoto the disulfide linkage are nitro, halo. hydroxyl, phosphate, sul

fonate, and the like. Examples of substituents which may permissiblyoccur in one or more of the positions etato the said disulfide linkage,that is to say, the positions numbered 3,5,3, and 5 in the said skeletalformula, include electrophilic radicals generally, and in particular,alkyl radicals containing from 1 to 18 carbons, alkoxy containing from 1to 18 carbon atoms, carboxy, nitro, phenyl, phenylalkyl containing from1 to 18 carbon atoms in the alkyl moiety thereof, carboxyalkylcontaining from 1 to 18 carbon atoms, and hydroxy alkyl of which thealkyl moiety contains from 1 to 18 carbon atoms. Also to be understoodas among the reagents to be employed in practicing the present inventionare the salts including particularly the alkali metal salts, alkaliearth metal salts, ammonium and substituted ammonium salts inclusive ofprimary, secondary, and tertiary alkyl and aryl amine salts and thealkyl and aryl esters of such of the compounds as contain carboxylgroups.

While methods of preparation of the present reagent compounds may, insome instances, lead to the production of compounds bilaterallysymmetrical as to the disulfide linkage, having identical substituentsin the same relative position on each of the benzene nuclei, suchsymmetry is not essential to the utility of the present reagentcompounds in the practice of the present test.

Among the reagents specifically to be employed in the conduct of testsof the present invention are bis(4- nitrophenyl) disulfide,bis(3-lauryl-4-nitrophenyl) disulfide, bis(3-carboxy-4-nitrophenyl)disulfide, bis(3-tertiarybutyl4-nitrophenyl) disulfide,bis(3-isopropoxy-4- nitrophenyl) disulfide, bis(3 phenyl-4-nitrophenyl)disulfide, bis(2-methoxy-4-nitrophenyl) disulfide, bis(3- carboxy-S-lauryloxyl-nitrophenyl) disulfide and, expressly, the sodium saltsprepared by reacting a sodiurn alkali compound such as sodium carbonate,bicarbonate, hydroxide, phosphate, or bisphosphate with each of theforegoing reagent compounds in which appears one or more carboxylgroups, whereby sodium replaces the hydrogen of at least part of thecarboxyl groups in each molecule of such compound. The more preferredreagents include bis(2-carboxy-4-nitrophenyl) disulfide,bis(3carboxy-4-nitrophenyl) disulfide and bis(4-nitrophenyl) disulfide.The most preferred of all the compounds of the said class isbis(3-carboxy-4-nitrophenyl) disulfide, together with, of course, itssalts and esters.

In carrying out the sulfhydryl tests, such as the qualitative tests ofthe present invention, at least one of the compounds of the group hereinset forth to be used as reagents is brought into contact with a materialof which the sulfhydryl content is unknown, thus forming a sulfhydryltest composition, wherein, when sulfhydryl groups are present, areaction characteristic of the present test takes place, and produces ayellow coloration in the resulting reaction mixture. The fact of thedevelopment of such color constitutes the qualitative positive responseof the present test. When under the said conditions, test reagent ispresent in such amount that addition to the test composition of furthertest reagent occasions no change in color, the intensity of the yellowcoloration is proportional to the concentration of sulfhydryl groups, asthe quantitative response to be estimated in the conduct of quantitativetests of the present invention. Depending upon such factors as samplesize, foreign colored substances present if any, the manner ofexhibition of the test compound according to the present invention,thickness of sample as measured along an optical axis under conditionsunder which the test is to be read, and like factors, the noveldisulfide reagent and sulfhydryl bearing material to be tested may bepresent within very wide limits of concentration. The reagents accordingto the present invention may be employed by dissolving them, with thesubstance to be tested, in a mutual solvent. Alternatively, a reagentaccording to the present invention may be dispersed on a member whichprovides mechanical support, such as absorbent paper, as by theevaporation thereupon of a solution of the present compounds in avolatile solvent such as a chlorinated hydrocarbon, or precipitation insuch paper of an insoluble salt or the like by reaction therein, with,for example, an alkaline earth compound and a carboxyl-substitutedreagent compound of the present invention, whereby to prepare sulfhydryltest papers. These papers may then be employed in the conduct of a testaccording to the present invention, for example by dipping, exposure tovapors, dusts, or gases, or in chromatographic techniques, whereby theyare contacted with a sulfhydryl-containing unknown. It is contemplatedthat such paper will usually be white before employment in such test,and will, correspondingly, be yellow upon completion of the test.However, such paper may be colored, either uniformly in in smalldiscrete colored areas, as, for example, a pale red whereby the development of the present test coloration causes it to turn to an apparentorange color; or, alternatively, such paper may be blue, whereby thedevelopment of the coloration according to the present test causes it toappear to be green. Many other such color combinations will be apparent.Moreover, the test compounds according to the present invention may bedispersed in or on a white, colored, or colorless substance which may bea solid or a granulated or finely subdivided material, such as silicagel, crushed fired ceramic material, and the like, which may then beemployed in a column. Any or all of these exhibitions of the testcompound according to the present invention may be estimated by, forexample, visual comparison of a test preparation against a knownstandard which known standard may be a comparable test preparation towhich has been added compositions or a composition containing a knownquantity of sulfhydryl groups, whereby to develop a yellow color ofintensity representing the said known. Alternatively, a color comparisonstandard may be prepared by printing with printing ink of selected coloron paper or the like; or such standard may be of colored glass, ceramic,or plastic or the like substance, 21 color of which is selected to matchor closely approach the color of a known sulfhydryl test color. It isnot necessary in the conduct of the tests according to the presentinvention that human visual response to the color yellow be observed, ormeasured; satisfactory estimations either qualitative or quantitative ofthe response of the reagents of the present test to sulfhydryl groupsmay be had by measurement of transmission of light at a specified wavelength or, alternatively, the absorption of light of complementarywavelengths. Such absorption may be measured as a diminution of thetotal amount of light transmitted, preferably in those wavelengths whichare, in the chromatic sense, complementary to the frequency of thetransmission maximum; or in alternative methods such as themicro-measurement of heating of preparations according to the tests, asa result of the absorption therein of the energy of incident,non-transmitted light. In other procedures the coloration characteristicof the practice of the test according to the present invention may beassayed, estimated, or made of reference by spectrophotometric and likemeans. Such mechanical optical means may rely upon the fact that thecolor developing in the test solution according to the present inventionpresents a light absorption maximum at the wavelength of 412millimicrons. The results of mechanicooptical measurement of the testresponse may be presented as an absorbance or transmittance factor, as aspectrogram, or, in equipment which refers the test coloration to aknown standard and, by optical means, makes a comparison, may beexpressed directly in concentration. Such concentration may be expressedvariously as percentage,

parts per million, or, in the case of single sources of sulfhydrylgroups, as a quantitative analytical result in terms of the compoundsource.

The test according to the present invention may be carried out in thepresence of substances having extraneous or anomalous color. When thetest is to be carried out in the presence of one or more of suchsubstances, 1t is often possible to prepare a known standard orstandards for reference wherein the resulting yellow sulfhydryl testcoloration is developed in the presence of the same anomalous orextraneous colorant as is present in the unknown so that referencethereto makes possible the direct sulfhydryl estimation of the unknownin the presence of the non-test color. Alternatively, the anomalouscolorant in a separate transparent container may be placed in theoptical system with a standard which does not contain the anomalouscolor, whereby the algebraically additive optical resultant exhibits theapproxb mate absorbance and appearance of the test standard as developedin the presence of such colorant.

It is among the desirable and useful properties of the reagentsaccording to the present invention, particularly but not exclusively asthey are dispersed on a chemically inert supporting solid, that the testresponse coloration developed over a period of time as, for example, byexposure to gases or dusts, represents the total cumulative or additivevalue of the sulfhydryl groups to which such test substance is exposed.Therefore, not only can the present tests be carried out in relativelyshort periods of time as in determining the composition of definiteunknown mixtures, but also, if desired, a sulfhydryl test preparationmay be exposed continuously over an extended period of time tocontinuing and cumulative contact with sulfhydryl groups or compositionscontaining such groups, whereby the resulting color which develops oversuch extended period of time accurately indicates the amount ofsulfhydryl groups contacted in such cumulative exposure.

Yet another advantage of at least many of the reagents to be employed inthe test of the present invention is their limited toxicity to warmblooded animals whereby, if desired, such compounds together withdesired excipients and additives and the like may be administered orallyor by injection in useful amounts to such animals, whereby sulfhydryltests of, for example, the contents of the alimentary canal may beconducted in vivo. In such application, a compound may be selected forlow solu bility in both water and lipid substances and the resultingyellow coloration may, thereafter, be extracted and exhibited forestimation quantitatively or qualitatively from the excrement of suchanimal, employing as solvent, for example, diethyl ether. Alternatively,a highly watersoluble compound according to the present invention may beingested for the conduct of in vivo tests of sulfhydryl concentrationsof body fluids, by the subsequent examinations of such fluids.

When the sulfhydryl group to be tested is present in' an ionized oreasily ionized form, the test according to the present invention may becarried out in either the presence or absence of solvent. When thesulfhydryl group to be tested is present in a non-ionized form,provision must be made for it to enter ionically into the reaction ofthe present test. This may easily be accomplished by presenting the testreagent in a basic state as, for example, by the presence intimatelyadmixed with it of at least a small amount of slightly basic solvent,such as perhaps, an amount of such solvent equimolecular with the saidtest reagent; or, the reagent, if a carboxyl substituted reagent, may bepresented as the salt such as the alkali metal or the amine orsubstituted amine salt thereof. The water-free or practically Water-freetestmaterials such as solutions, columns, and papers as de-- scribed,wherein are included intimately admixed with the test reagent smallamounts of basic solvents, are consid-' ered to be part of the presentinvention; Suitable slightly" basic solvents include, among others,liquid amines, pyridine, and the like.

When either the reagent or the sulfhydryl-bearing composition is presentin a liquid phase, and especially when present in an aqueous phase, itis desirable for the eiiicient conduct of the present test that theliquid phase be maintained in a pH within the range of 5 to 9 andpreferably within the range of 7 to 8 during the time the test is to becarried out and estimated. At a pH below about 5, the color-producingreaction of the present test is inhibited, and the color developmenttakes place so slowly as to render the test inefficient. At a pH muchbelow 5 the development of the yellow coloration of the present test maytake place so slowly as to be imperceptible and worthless in laboratoryprocedures. At a pH maintained continuously in the range of 7 to 8during and after the conduct of the present test, the resulting yellowcoloration develops quickly, accurately, and is sufficiently stable forall ordinary laboratory uses. At a pH much greater than 9, the testreagent decomposes under the influence of hydroxide ion at a rate whichincreases as the pH increases much above 9. Thus, for example, yellowsolutions prepared in the conduct of the present test in the pH range of7 to 8 have proved to be completely stable in color for at least as longas a year, whereas similar solutions wherein the test is carried out ata pH in the range of 11 to 12 give the desired yellow colorationpromptly, but the resulting coloration disappears through decompositionor gives way to anomalous and meaningless color changes within a shorttime, such as an hour or less. When the present test reagents are to beemployed in solution such as aqueous solution, preferably such solutionshould contain buffering agents whereby to maintain the pH in thepreferred range of 7 to 8. This is especially true if such solutions areto be held in storage for a period of time prior to their use. If suchbuffering is not to be employed, fresh reagent solutions should beprepared for each test, and the test carried out preferably withcontinuous measurement of pH which pH is continuously maintained inappropriate manners in the preferred range of at least 5 to 9 andpreferably 7 to 8.

When carrying out the test of the present invention in non-aqueoussolution, water-free pyridine of such purity as to be essentiallycolorless is a satisfactory solvent. Alternatively, a colorlesshydrocarbon or halogenated hydrocaroon solvent may be employed in thepresence of at least a minimal amount, such as an amount equimolecularwith the test reagent, of an organic base such as a nitrogen-bearingcompound which may be pyridine or a primary, secondary, or preferablytertiary amine, free from water.

When the present test is carried out upon a sulfhydrylbearingcomposition in a solid phase, which may be dispersed in a non-solventliquid system, the resulting test coloration determines only thosesurficial sulfhydryl groups available to make contact with the presentreagent. In certain biochemical applications this may be a preferredmanner of carrying out the present tests. When it has been desired toestimate the total sulfhydryl content of a composition upon the basis ofa quantitative determination of sur'ficial sulfhydryls, it has oftenbeen possible to calculate a factor greater than unity which may becalled a form factor expressing the ratio between surficial sulfhydrylgroups and total sulfhydryl groups in such solids. The product frommultiplication of determined surficial sulfhydryl content by the saidform factor represents tota sulfhydryl content subject, of course, toprocedural error.

In carrying out the tests according to the present invention, the testreaction may be carried out at any temperature over a very wide range.In practice, conduct of the test at temperatures lower than 0 C. resultsin a very slow development of the test color; whereas temperatures muchabove about 50 C. may result in the thermal decomposition of the testcompounds. A preferred temperature range for the conduct of the presenttests is, therefore, from room temperature, that is to say about 20 (3.,to about 50 C.

In carrying out quantitative sulfhydryl estimations in the tests of thepresent invention, it is to be understood that preliminary orrange-finding qualitative or quantitative estimations may be carried outto ascertain the fact of the presence of, and the approximateconcentration in an unknown composition of the sulfhydryl groupstherein. For example, a dilute solution of a reagent of the presentinvention may be employed whereby to determine whether the unknowncontains sulfhydryl groups in quantity sufficient to saturate the colorresponse of such dilute test solution. Similarly, a more concentratedsolution of reagent of the present invention may be employed whereby todetermine whether the unknown contains sulfhydryl groups in quantitiessufiicient to saturate the color response of such concentrated testsolutions. In such manners, a general range of sulfhydryl concentrationmay quickly be ascertained, and thereafter, the present test may beemployed in any degree of precision desired in the measurement ofsulfhydryl groups within the range thus established. As but oneembodiment of the rangefinding use of the present tests, a test paper ofthe sort described may be prepared containing a relatively highconcentration of at least one of the reagents of the present invention,intended to be used for rough estimation and range-finding purposespreliminary to the conduct of more precise estimations of sulfhydrylgroups if such more precise estimations are desired.

The sulfhydryl test of the present invention is illustrated by thefollowing examples which are not to be considered as limiting.

EXAMPLE 1 Bis(4-nitrophenyl) disulfide (melting at 183-184" C.) wasdissolved in acetone to prepare a solution containing 30.8 milligrams ofthe disulfide compound per 100 milliliters. This solution was 1/ 1000molar and is referred to in the present example as the reagent solution.A 0.1 molar aqueous solution of disodiurn hydrogen phosphate and a 0.1molar aqueous solution of sodium dihydrogen phosphate were mixedtogether to form an aqueous solution having a pH value of 8.0. Thissolution is referred to in the present example as the buifer solution.In carrying out the sulfhydryl estimation, a measured amount of thesulfhydryl-bearing unknown was dissolved in 4 milliliters acetone. Onemilliliter each of the reagent solution and of the butler solution wereadded to the said unknown. Thereupon a color change took place, wherebythe resulting mixture appeared to be yellow. The light absorbance of theyellow mixture as referred to a corresponding blank solution is measuredat 412 millimicrons wave length of transmitted light. The concentrationof the sulfhydryl group in the final solution is calculated from theformula wherein C is the concentration of the sulfhydryl group and A=2log T when T is the transmission of light through the colored unknownexpressed as a percentage of light transmitted by the correspondingblank. Such values of A are commonly read directly from photometers ofknown kinds. When such test is carried out with simple mercaptans suchas the lower alkyl mercaptans, the 'test reaction goes to completionvirtually instantaneously and the resulting color remains stable for atleast 0.5 hour. For cysteine and related mercaptoethylarnines, fullintensity is developed after 60 to minutes. To the eye, the developmentof the test appears as a change from a colorless or nearly colorlesssolution to a vivid and intense yellow to yellow-orange solution whichmay be Table I Known Amount Amount Meas- Amount Measof Tested Comured bythe med as Percent- Compound Tested pound Added Test of the Known(Micrograms Present In- Amount Added per 4 ml.) vention 20. 1 20. 2 10120.1 20.1 20.1 20. 2 2-Mereaptoethane 1005 10. 2 ml 10.05 10.0 99. 10.059.9 98. 5 5.03 5. 1 101 5.03 5.3 9799 r 5. 03 4. 5 ys L .9 82 1.10 1. 091 1. 10 1. 2 100 limit of concentration of tested compounds in testsolutions proved to be approximately 2 to 4X10 molar, in which range theresulting color characteristic of the test was of such dilution as toreach the practicable lower limit of the sensitivity of the photometer.Qualitative sulfhydryl tests have proved to be much more sensitive.

EXAMPLE 2 LHL'LSUREMENT OF SULFHYDRYL 'COl\".l?EZ\"ll IN \VHOLE BLOODThis example illustrates the conduct of the test of the presentinvention in the presence of materials which alter optical properties ofthe test coloration, as, by the presence of non-test colorants.

A mixture is prepared containing 0.01 milliliter whole blood, 5milliliters water, and 2.7 milliliters acetone. To this is added 2milliliters of the phosphate buffer solution described in Example 1. Theresulting buffered blood solution is allowed to stand at roomtemperature for 1 hour. Thereafter, 0.3 milliliter of the reagentsolution of bis(4-nitrophenyl) disulfide as described in Example 1 isadded to the buttered blood solution and thoroughly stirred and theabsorbance at the 412 millimicron wave length is photometricallydetermined, wherefrom is calculated the sulfhydryl content of the blood.As a blank to be employed in the photometer, for the absorbance of theblood solution alone, the following preparation is employed; 0.010milliliter blood, 5.0 milliliters water, 2.0 milliliters butter and 3milliliters acetone. This preparation is allowed to stand at roomtemperature, along with the test preparation to which the reagent is tobe added.

Of the blood samples tested in this method, most reached equilibriumresponse in 0.5 minute to 1 minute after addition of the reagentsolution to the buffered blood sample. However, in occasional samples aslong as 40 minutes were required to reach equilibrium. Therefore, insuch work, it is desirable to measure absorbance successively at severaldifferent times noting any change. When two or more successive readingsare alike, equilibrium may be assumed to have been reached. Samples ofblood tested and results of such tests are indicated in the followingtable:

BLOOD lVlERCAPTAN CONCENTRATIONS Individual [torn Whom Sample was TakenThe individuals represented in the above test were male except for thelast two who were females.

When it is desired to estimate the sulfhydryl concentration of bloodserum, the following preparation may be employed; the first absorbancemeasurement should be taken immediately upon completion of thepreparation. As a test solution, 0.1 milliliter serum, 1.0 milliliter ofthe phosphate buffer described in Example 1, and 0.3 milliliter of thedisulfide reagent solution described in Example 1, together with 2.7milliliters acetone and 5.9 milliliters Water. As a blank preparationfor comparison, 0.1 milliliter blood serum, 1.0 milliliter buffersolution, 5.9 milliliters Water, 3.0 milliliters acetone.

EXAMPLE 3 Bis(3-carboxy-4-nitrophenyl) disulfide, sodium dihydrogenphosphate and disodium hydrogen phosphate were dissolved in water toform a phosphate buffered aqueous solution thereof containing oneone-thousandth grammole of phosphate and one ten-thousandth gram-mole ofdisulfide compound (39.6 milligrams) in milliliters of the solution, theultimate solution having a pH of 8.0 This solution will be referred toin the present example, and in others carried out in similar manner, asthe reagent solution.

A supplementary solution was prepared by mixing together a 0.1 molaraqueous solution of disodium hydrolgBH phosphate and a 0.1 molar aqueoussolution of sodium dihydrogen phosphate to form an aqueous solutionthereof having a pH of 8. This solution will be referred to in thepresent example and others carried out in similar manner as the buttersolution.

In carrying out the sulfhydryl estimation, :1 measured amount of the sulfhydryl-bearing unknown is initially dissolved in 8 milliliters of Wateror in a waterrniscible solvent. One milliliter each of the reagent andof the buffer are added to the unknown solution. If a water misciblesolvent is used to dissolve the unknown, it may be necessary, accordingto this test procedure, to add further Water so that the resulting testmixture has a volume of 10 milliliters. The resulting mixture isthoroughly agitated and allowed to stand at room temperature for atleast one minute and not more than four hours. A yellow color having alight absorption maximum at 412 millimicrons wave length develops in thetest mixture, which color is of an intensity that is directlyproportional to the amount of sulfhydryl groups present over the rangeof from approximately 3.7 millionths to 73 millionths gram-mole perliter. The color can be measured in any standard colorimeter orphotometer or may be visually estimated in comparison with standardscontaining known concentrations of sulfhydryl groups and reagent,prepared in similar manner. When sulfhydryl estimation is to be carriedout in the presence of colored materials whose colors alter the yellowappearance or the characteristic absorbance of the test, viewing ormeasurement against a known standard may be made with a suitableconcentration of the colored unknown in the optical axis of and beforesuch standard. Thus, the test has proved to be useful in the presence ofsuch colored materials as tissue extractives, urine, blood, and thelike.

Tests conducted on tissue extracts which were made by extracting thetissues with hot 80 percent ethanol gave the results set forth in thefollowing table.

TISSUE SULFHYDRYL ANALYSIS Sull'hydryl Content as Millimoles per Gram of80 percent Hot Ethanol Tissue Extract Time to Develop Full Tissue(Rabbit) Color (minutes) 1. Less than one minute. 2.

EXAMPLE 4 EXAMPLE 5 A solution of crude soap is adjusted to pH 8 and thesul'fhydryl content thereof determined using as test reagent a bufferedsolution of bis(3-lauryl-4-nitrophenyl) disulfide. This reagent ischosen for the similarity of its solubility performance to that of thesaid soap.

EXAMPLE 6 A sample of coal is crushed and then ground in pyridine toyield a paste suspension containing the said coal as an impalpablepowder. The sulfhydryl concentration in the resulting suspension isdetermined in manners similar to the determination in the foregoingexperiments, employing a photometer cell designed to exhibit the testsuspension in one tenth millimeter section, and employing, as reagent,the compound bis(3-phenyl-4-nitrophenyl) disulfide, dissolved inpyridine.

EXAMPLE 7 The sulfhydryl content of an unrefined sour, that is to say,sulfur-bearing, crude petroleum is determined by diluting the crudepetroleum with petroleum ether and employing, as reagent,bis(3-carboxy-5-lauryloxy-4- nitrophenyl) disulfide; and using a similardilution as a color-reference blank.

EXAMPLE 8 The dilution of sulfhydryl-bearing waste outflow from a wastedisposal plant into a natural body of water is automatically controlledby the simultaneous photometric scanning of, on the one hand, acontinuously replaced sample from the said natural body of watercontaining the diluted waste, into which sample is continuously meteredand intimately mixed a reagent according to the present inventiontogether with buffering agents and, on the other hand, a color standardcorresponding to an acceptable level of sulfhyclryl content. Theelectrical outputs of the photometric systems are connected inopposition one to another through a resistance bridge and the system sobalanced that a current flow in the output of the bridge networkcorresponds to a sulfhydryl concentration at about a maximum tolerablelevel in the diluted output. Such current flow is caused to actuate areversible solenoid-controlled valve and alarm system by the action ofwhich sulfhydryl-bearing waste outflow is interrupted and an alarm isenergized until, responsive ot a reduced sulfhydryl content, the bridgeoutput current ceases to flow and the solenoid-controlled valve operatesin reverse whereby the outflow is resumed.

EXAMPLE 9 A wet-strength absorbent paper is impregnated in a moistcondition with calcium bicarbonate and there is subsequently introducedinto the same paper an excess of bis(3-carboxy-4-nitrophenyl) disulfidewhereby the waterinsoluble calcium salt of the said disulfide compoundis formed in situ within the said paper. Thereafter, excess disulfideand calcium bicarbonate materials are removed by washing with water andthe resulting paper dried.

Another test paper is prepared by soaking a wet-strength absorbent paperin a buifered solution of bis(2-methoxy-4- nitrophenyl) disulfide, andthe paper is thereafter evaporated to dryness.

In similar manner, a granular apparently dry silica gel is immersed inan isopropanol solution of bis(3-isopropoxy-4-nitrophenyl) disulfide,the excess is removed, and the silica gel bearing a surface applicationof the said disulfide is gently warmed to vaporize and remove solvent.

In each of the foregoing preparations, the evaporation to dryness of theresulting test agent is carried out in an atmosphere essentially freefrom sulfhydryl groups and at temperatures not exceeding 56 C. Theproducts of the said preparations are subsequently used for quantitativesulfhydryl estimation in gases and liquids, by comparison of a testpaper brought into contact with the unknown with a similar test paperbrought into contact with a known sulfhydryl solution. Each of the testmaterials is essentially white in color before being used in such testand during the course of the said test develops a yellow color whoseintensity depends upon the concentration of sulihydryl groups.

In each of the foregoing examples, and in other similar preparations ofthe compounds to be used in tests according to the present invention,the preparation of a reagent solution containing one ten-thousandthgram-mole of disulfide reagent compound in milliliters of solutionobtains a reagent solution wherein the yellow color developing in theconduct or" the sulfhydryl test is directly proportional to the amountof the sulfhydryl groups present over approximately the range of from3.7 millionths to 73 millionths gram-mole per liter. The test reactionand the development of yellow coloration having a light absorptionmaximum at a wave length of 412 millimicrons proceeds reliably andquantitatively over a wide range of concentrations of reagent andreagent solution, being limited only by, at the lower extreme ofconcentration, the limit of sensitivity of the eye of the observer, oralternatively, of the mechanico-optical system wherein the lightabsorbance is measured; and at the other extreme of concentration, bythe limits of the powers of the observer, or the development of acoloration so intense as to exceed the limits which can be measured inthe photometric equipment employed. In practice, however, these limitsdo not essentially restrict the range of sulfhydryl group concentrationswhich can be tested, inasmuch as sulfhydryl-bearing materials may bediluted or concentrated by dissolving or dispersing them in a suitableliquid in known ratio, such ratio being chosen to exhibit the resultingsulfhydryl concentration in a range which can conveniently be estimatedin the quantitative method according to the present invention, thesulfhydryl concentration of the original being thereafter easilycalculated. Also, the colored test solution may be viewed in comparisonwith a standard, both test and standard being controlled as to depth ofviewed section.

One of the compounds to be employed as reagent in conduct of the presenttest is known, although its use in such test is novel.Bis(4-nitrophenyl) disulfide is set forth in Beilstein, Handbuch derorganischen Chemie at 6, 340; I, 160; and II, 312. This compound may bedissolved in acetone, and in such solvent has been successfully employedin the conduct of the qualitative test according to the presentinvention. However, in quantitative tests, especially those conductedwith the assistance of a photometer, the results obtained by the use ofthe said compound proved to be unreliable. Attempts to replicate testresults successively by the testing of aliquot portions of the same testsolution gave anomalous results. This was especially noted in theconduct of sulfhydryl tests on substances naturally occurring in livingorganisms, including, for example, proteins. Unexpectedly, certain othercompounds of the class set forth to be used as reagents according to thepresent tests have been used successfully, overcoming the saidditficulties and other difficulties encountered in the use of thebis(4-nitrophenyl disulfide. The novel compoundsbis(3-carboxy-4-nitrophenyi) disulfide and bis(2-carboxy-4-nitrophenyl)disulfide were found to give reliable and replicable test results.

The following example, which is not to be considered as limiting, setsforth one method by which may be prepared thebis(3-carboxy-4-nitrophenyl) disulfide.

EXAMPLE In the first step of the process, six grams (0.0335 mole) of5-chloro-2-nitro-benzoic acid was suspended in 150 milliliters of waterto which concentrated aqueous sodium hydroxide was added with constantstirring to bring the mixture to a pH of 7.2 and dissolve the5-chloro-2- nitro-benzoic acid as a sodium salt. Sodium sulfide (Na SBHO, 12 grams: 0.050 mole) dissolved in milliliters of water was addedportionwise thereto with continuous stirring. The reaction mixture thusformed was heated at 50 C. for 1.25 hours with continuous stirring.Stirring was then discontinued and the reaction mixture allowed to cool.When it had cooled to approximately room temperature the reactionmixture was acidified by the addition of concentrated hydrochloric acidto a pH of approximately 1 whereupon an oily intermediate productseparated. The oily product was removed in a separatory funnel. In thesecond step of the process, the said oily intermediate product wasdissolved in 50 milliliters of water by the addition of concentratedaqueous sodium hydroxide to a pH of 7.2. To this solution approximately30 milliliters of an aqueous solution of iodine in 5 weight percentconcentration and potassium iodide in a 2 weight percent concentrationwas added dropwise and with continuous stirring over a period of 15minutes at room temperature. The quantity of the aqueous solution ofiodine and potassium iodide to be added was determined by color changein the resulting mixture. When addition of the iodine solution began,the mixture to which it was added was of a deep orange-yellow color.During the course of the addition this color became li hter andeventually changed to a pale yellow. Further additions of the iodinesolution tinged the reaction mixture with the reddish-brown color ofiodine. At this point, addition of iodine solution was discontinued. Thepale yellow reaction mixture thus formed was then acidified to a pH ofapproximately 1 by the addition of concentrated hydrochloric acid, toproduce the desired bis(3-carboxy-4-nitrophenyl) disulfide product. Theproduct was separated from the reaction mixture by repeated extractionswith diethyl ether. Ether was evaporated from the extract and theresidual product was purified by recrystallization from glacial aceticacid to obtain a bis(3-carboxy-4-nitrophenyl) disulfide product as apale yellow crystalline solid which was slightly soluble in each of thesolvents ether, alcohol, acetone, and toluene and was insoluble inwater, but readily soluble in dilute aqueous alkali. The product 12melted with decomposition at 237 to 238 C. Product solutions exhibited acharacteristic ultraviolet absorption spectrum with a peak at 315millimicrons. Titration of one gram-mole of the purified productrequired 2 gram chemical equivalents of base.Bis(3-carboxy-4-nitrophenyl) disulfide has a molecular weight of 396.5.

In similar preparation there is prepared a novel bis(2-carboxy-4-nitrophenyl) disulfide, by employing as starting reactant2-chloro-5-nitrobenzoic acid. The bis(2-carboxy-4-nitrophenyl) disulfideis useful in a manner similar to the bis(3-carboxy-4-nitrophenyl)disulfide but exhibits numerous unexpected distinctive and differentproperties in use. As but one example of such distinctive properties,the 2-carboxy-4-nitrophenyl compound reacts at a much slower rate,requiring approximately a half hour to develop color responses which the3-carboxy compound usually develops in less than a minute.

The said novel bis(2-carboxy-4-nitrophenyl) disulfide is especiallyuseful in the conduct of automatically controlled operations such asthat set forth in Example 8, in use of apparatus wherein a sensingapparatus is employed, whereby the activation of a response such as theoperation of a solenoid-controlled device is delayed until a replicatenumber such as two or more sensings of the contents of a sulfhydryl testcell are found lie within predetermined limits of equivalents. By theemployment of the slowreacting Z-carboxy compound it is possible toachieve, as results of a small number of tests, control responses whichapproximate an integration of a larger number of shorttime tests andavoid sudden and short-cycle operations of the controlled device.Alternatively, when it is desired to operate such equipment in a mannerto obtain very quick response to sulfhydryl content as measured, the2-carboxy substituted compound should be avoided, and, for example, the3-carboxy compound employed instead. Also, mixtures of the two suchcompounds may be prepared, which mixtures react, in a desired degree ofapproach to completeness, in known times, with the result that theoperative cycle of automatic equipment may be adjusted by such chemicalmeans, within wide limits.

EXAMPLE 11 The novel compound bis(3-lauryl-4-nitrophenyl) disulfide isprepared. In one manner of preparation, laurylbenzene is nitrated by theaction of nitric acid in the presence of sulfuric acid, whereby thereare produced mixed isomers of ring-nitrated laurylbenzene. Such mixtureis resolved into its components from which the o-nitrolauryl benzene isseparated by crystallization from hot alkanol solution as such solutioncools. The o-nitrolaurylbenzene is then dissolved in carbontetrachloride and, in the resulting solution, chlorinated by theaddition thereto of gaseous chlorine in an amount approximatelyequimolecular with the o-nitrolauryl benzene. Upon completion of thechlorination, the chlorinated mixture is purged with nitrogen, to removeunreacted chlorine and hydrogen chloride of reaction and the resultingsolution is thereafter dried over anhydrous magnesium sulfate andtreated by the addition thereto of sodium sulfide and sulfur as finelydivided solids. Such addition is carried out slowly, portionwise, andwith vigorous stirring at room temperature. Stirring is thereaftercontinued for a period of time to carry the reaction to completion andform the desired bis(3-lauryl- -nitrophenyl) disulfide reagent which maythereafter be extracted from the resulting reaction mixture by evaporating the chlorinated hydrocarbon solvent nearly to dryness under reducedpressure and with gentle heating to temperatures not in excess of 50 C.,and thereafter extracting the desired disulfide compound with petroleumother as extractive solvent. Bis(3-lauryl-4-nitrophenyl) disulfide has amolecular weight of 644.8. In manner similar to the foregoing there isprepared a bis(3-phenyl-4-nitrophenyl) disulfide compound by theemployment, as starting material, of biphenyl.Bis(3-phenyl-4-nitrophenyl) disulfide has a molecular weight of 461.5.

1? EXAMPLE 12 The novel compound bis(3-isopropoxy-4-nitrophenyl)disulfide is prepared by a two-step process. The first such stepconsists of the chlorination of bis(4-nitrophenyl) disulfide by thedirect addition of gaseous chlorine to the said disulfide compounddissolved in a chlorinated hydrocarbon solvent, purging the resultingreaction mixture with nitrogen to remove unreacted chlorine and hydrogenchlorine of reaction, drying the resulting chlorinated mixture. Thesecond step consists of adding to the dried first step product aquantity of sodium isopropoxide approximately equimolecular with thestarting disulfide compound. The isopropoxide is added slowly,portionwise, and with stirring over a period of time to carry thereaction to completion. The resulting compound may thereafter readily beseparated in such manners as fractional crystallization, and the like.Bis(3-isopropoxy-4-nitrophenyl) disulfide has a molecular weight of424.9.

EXAMPLE 13 The compound bis(2-methoxy-4-nitrophenyl) disulfide is novel.in its preparation, there is employed, as starting reaction the compound3-nitroani-sole which is set forth in Beilstein, Handbuch, at 6, 224; I,116; and II, 214. The anisole compound is directly chlorinated, thechlorination products purged with an inert gas and subsequently driedand treated with a sulfur-sodium sulfide mixture. Thereafter, thedesired bis(2-methoxy-4-nitrophenyl) disuifide compound is separated bysolvent extraction employing methanol as solvent. Thebis(2-methoxy-4-nitrophenyl)- disulfide has a molecular weight of 368.3.

EXAMPLE 14 The novel compound bis(3-carboxy-5-lauryloxy-4-nitrophenyl)disulfide is prepared. In such preparation there is employed, asstarting reactant, the bis(3-carboxynitrophenyl) disulfide whosepreparation is set forth in Example 11. The said starting reactant ischlorinated by direct addition of chlorine in an amount equimolecularwith the disulfide compound, in a chlorinated hydrocarbon solvent. Theresulting mixture is purged with an inert gas and dried and thereafterreacted with sodium lauryloxide and the desired product thereafterseparated by fractional crystallization, from a mixture of petroleumether and chlorinated hydrocarbon as solvents. Bis(3-carboxy-5-lauryloxy-4-nitrophenyl) disulfide has a molecular weight of 733.0.

In manners similar to the foregoing preparations, other reagentcompounds of the present invention are prepared.

The present application is a continuation-in-part of application SerialNo. 712,667, filed February 3, 1958, now abandoned.

I claim:

1. A method of testing for su'lfhydryl groups in an unknown suspected ofcontaining sulfhydryl groups which comprises the step of bringing thesaid unknown into contact with a test reagent compound containing thestructure corresponding to the skeletal formula substituents thereuponbeing limited in that any substituent in a position metato a nitro groupis non-nucleophilic, except that carboxyl groups and salts thereof mayoccur in such position, and any branched substituent in such positionmust have its branching moieties separated from the benzene nucleus byat least one atom; and thereafter comparing the resulting yellowcoloration in the resulting test reaction mixture with at least onecolor standard.

2. The method of claim 1, modified by the limitation that the testreagent compound is employed in such amount that bringing the testreaction mixture of claim 1 into contact with further test reagentcompound does not increase the depth of the resulting reagent mixturecolor.

References Cited in the file of this patent UNXTED STATES PATENTSHaddock Jan. 7, 1936 Gowberg Dec. 2, 1958 OTHER REFERENCES

1. A METHOD OF TESTING FOR SULFHYDRYL GROUPS IN AN UNKNOWN SUSPECTED OFCONTAINING SULFHYDRYL GROUPS WHICH COMPRISES THE STEP OF BRINGING THESIAD UNKNOWN INTO CONTACT WITH A TEST REAGENT COMPOUND CONTAINING THESTRUCTURE CORRESPONDING TO THE SKELETAL FORMULA